Colorectal cancer is a leading cause of cancer-related deaths in the United States and is a major cause of morbidity and mortality among the US Veterans. While early stages of colorectal cancer are highly curable by surgical resection, the prognosis of patients with metastatic disease remains grave. Promising targeted colorectal cancer treatments, including inhibitors of vascular endothelial growth factor and monoclonal antibodies against epidermal growth factor receptor have resulted in significant improvement of overall survival in patients with metastatic disease; however, the relatively transient or individually restricted nature of clinical responses o currently available targeted therapies underlines the urgency for further development of novel therapeutic strategies that specifically target therapy-resistant cancer cell populations, which may coincide with cancer stem cells. Multiple studies have demonstrated the existence of cancer stem cells in human colorectal cancer and their contribution to colorectal cancer metastatic progression and therapeutic resistance. Thus, specific targeting of cancer stem cells could provide for a novel strategy to eradicate cancers currently resistant to systemic therapy. ABCB5 is a novel human multidrug resistance mediator recently shown by the applicant in the first funding period of this proposal to be expressed in therapy-resistant colorectal cancer stem cells and to correlate with clinical cancer progression (Wilson et al. Cancer Res. 2011). Moreover, this landmark study, the first on the critical role of ABCB5 in colorectal cancer and recently independently confirmed by other investigators in the field (Kugimiya et al. J Cell Mol Med. 2015), demonstrated that ABCB5 blockade leads to inhibition of tumor growth and invasion, and sensitizes colorectal cancer to 5-FU-induced cell killing. In additional ground- breaking results during the initial project funding period, the applicant recently identified a criical role of ABCB5 in normal stem cell maintenance through a novel anti-apoptotic function involving p53 stabilization (Ksander et al. Nature 2014) and has now in further preliminary results also established this critical ABCB5 function in colorectal cancer stem cells. Based on these findings we hypothesize that specific targeting of ABCB5-expressing colorectal cancer stem cells has the potential to result in eradication of disseminated disease currently resistant to conventional therapies. We therefore propose in this project to (i) Dissect ABCB5 molecular roles in colorectal cancer stem cell maintenance, colorectal cancer stem cell-dependent tumor initiation and metastatic progression, and colorectal cancer stem cell therapeutic resistance in novel disease models in which ABCB5 function is genetically ablated; (ii) Establish colorectal cancer stem cell therapeutic responses to approved or novel emerging colorectal cancer treatment modalities, utilizing novel ABCB5 reporter mouse models of genetic and inflammation-driven intestinal tumorigenesis; and (iii) Target ABCB5-positive colorectal cancer stem cells in a translationally relevant manner in human-to-mouse xenotransplantation models singly or in novel combination strategies using newly generated fully human high-affinity anti-ABCB5 monoclonal antibodies (Ksander et al. Nature 2014). The proposed studies will further establish the clinical relevance and therapeutic importance of ABCB5 in colorectal cancer and should pave the way to successful targeting of ABCB5_positive colorectal cancer stem cells in human patients for improved clinical therapy.